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Plasmid DNA Purification User manual NucleoBond® Xtra Midi NucleoBond® Xtra Maxi NucleoBond® Xtra Midi Plus NucleoBond® Xtra Maxi Plus

October 2006/Rev. 02 www.mn-net.com

MACHEREY-NAGEL

MN MACHEREY-NAGEL

EN ISO 9001:2000 CERTIFIED

MACHEREY-NAGEL MACHEREY-NAGEL MACHEREY-NAGEL

MN

Protocol-at-a-glance (Rev. 02) Plasmid DNA Purification (NucleoBond® Xtra Midi / Maxi) Midi

1

Cultivate and harvest bacterial cells

2

Cell lysis

3

Maxi

4,500-6,000 x g 15 min at 4°C high-copy / low-copy

high-copy / low-copy

Buffer RES

8 ml / 16 ml

12 ml / 24 ml

Buffer LYS

8 ml / 16 ml

12 ml / 24 ml

Equilibration of the column together with inserted column filter

4

Neutralization

5

Clarification and loading of the lysate

Buffer EQU 25 ml

Buffer EQU 12 ml

Buffer NEU 8 ml / 16 ml

Buffer NEU 12 ml / 24 ml invert the tube 3 times

load lysate on NucleoBond® Xtra column filter lysate is simultaneously cleared and loaded onto the NucleoBond® Xtra column

6

1st Washing

7

Discard NucleoBond® Xtra column filter

8

2nd Washing

9

Elution

10 Precipitation

11 Wash and dry DNA pellet

12 Reconstitute DNA

Buffer EQU 5 ml

Buffer EQU 15 ml

discard NucleoBond® Xtra column filter

discard NucleoBond® Xtra column filter

Buffer WASH 8 ml

Buffer WASH 25 ml

Buffer ELU 5 ml

Buffer ELU 15 ml

NucleoBond® Xtra Midi

NucleoBond® Xtra Midi Plus

NucleoBond® Xtra Maxi

NucleoBond® Xtra Maxi Plus

Isopropanol 3.5 ml

Isopropanol 3.5 ml

Isopropanol 10.5 ml

Isopropanol 10.5 ml

15,000 x g 30 min at 4°C

load NucleoBond® Finalizer

15,000 x g 30 min at 4°C

load NucleoBond® Finalizer Large

70% ethanol 2 ml

70% ethanol 2 ml

70% ethanol 5 ml

70% ethanol 5 ml

15,000 x g 5 min at RT

/

15,000 x g 10 min at RT

/

5-10 min


3 x air

10-15 min


3 x air

Appropriate volume of TE buffer

Buffer TRIS 500-1000 µl

Appropriate volume of TE buffer

Buffer TRIS 500-1000 µl

MACHEREY-NAGEL GmbH & Co. KG • Neumann-Neander Str. 6-8 • D-52355 Düren • Germany Tel.: +49 (0) 24 21 969 270 • Fax: +49 (0) 24 21 969 279 • e-mail: [email protected]

Plasmid DNA Purification

Table of contents 1 Kit contents

5

2 Kit specifications

7

3 About this user manual

8

4 NucleoBond® Xtra plasmid purification system

9

4.1 Basic principle

9

4.2 NucleoBond® Xtra anion-exchange columns

9

4.3 Growth of bacterial cultures

11

4.4 Culture volume for high-copy plasmids

12

4.5 Culture volume for low-copy plasmids

13

4.6 Cell lysis

14

4.7 Difficult-to-lyse strains

14

4.8 Setup of NucleoBond® Xtra columns

15

4.9 Filtration and loading of the lysate

16

4.10 Washing of the column

16

4.11 Elution and concentration of plasmid DNA

17

4.12 Determination of DNA yield and quality

19

4.13 Convenient stopping points

19

5 Storage conditions and preparation of working solutions

20

6 Safety instructions - risk and safety phrases

21

7 NucleoBond® Xtra plasmid purification

22

7.1 High-copy plasmid purification (Midi, Maxi) - English

22

7.2 Low-copy plasmid purification (Midi, Maxi) - English

27

7.3 Concentration of NucleoBond® Xtra eluates with the NucleoBond® Finalizers - English

30

7.4 High-copy plasmid purification (Midi, Maxi) - German

32

7.5 Low-copy plasmid purification (Midi, Maxi) - German

38

7.6 Concentration of NucleoBond® Xtra eluates with the NucleoBond® Finalizers – German

42

7.7 High-copy plasmid purification (Midi, Maxi) - French

44

7.8 Low-copy plasmid purification (Midi, Maxi) - French

50

MACHEREY-NAGEL – 10/2006/ Rev. 02

3

Plasmid DNA Purification 7.9 Concentration of NucleoBond® Xtra eluates with the NucleoBond® Finalizers – French 8 Appendix

4

54 56

8.1 Troubleshooting

56

8.2 Ordering information

63

8.3 Product use restriction / warranty

63

MACHEREY-NAGEL – 10/2006/ Rev. 02

Plasmid DNA Purification

1

Kit contents NucleoBond® Xtra Midi

NucleoBond® Xtra Midi Plus

10 preps

50 preps

100 preps

10 preps

50 preps

740410.10

740410.50

740410.100

740412.10

740412.50

Buffer RES

100 ml

500 ml

1000 ml

100 ml

500 ml

Buffer LYS

4 x 25 ml

500 ml

1000 ml

4 x 25 ml

500 ml

Buffer NEU

100 ml

500 ml

1000 ml

100 ml

500 ml

Buffer EQU

200 ml

2 x 500 ml

2 x 1000 ml

200 ml

2 x 500 ml

Buffer WASH

100 ml

500 ml

1000 ml

100 ml

500 ml

Buffer ELU

60 ml

300 ml

600 ml

60 ml

300 ml

RNase A
(lyophilized)

6 mg

30 mg

2 x 30 mg

6 mg

30 mg

NucleoBond® Xtra Midi columns

10

50

100

10

50

NucleoBond® Xtra Midi column filters

10

50

100

10

50

NucleoBond® Finalizer

-

-

-

10

50

30 ml Syringes

-

-

-

10

50

1 ml Syringes

-

-

-

10

50

Buffer TRIS

-

-

-

15 ml

75 ml

Plastic washer

5

10

10

5

10

Protocol

1

1

1

1

1

Cat. No.




For preparation of working solutions and storage conditions see section 5.

MACHEREY-NAGEL – 10/2006/ Rev. 02

5

Plasmid DNA Purification

1

Kit contents continued NucleoBond® Xtra Maxi

NucleoBond® Xtra Maxi Plus

10 preps

50 preps

100 preps

10 preps

50 preps

740414.10

740414.50

740414.100

740416.10

740416.50

Buffer RES

150 ml

750 ml

2 x 750 ml

150 ml

750 ml

Buffer LYS

150 ml

750 ml

2 x 750 ml

150 ml

750 ml

Buffer NEU

150 ml

750 ml

2 x 750 ml

150 ml

750 ml

Buffer EQU

500 ml

2 x 1000 ml 500 ml

5 x 1000 ml

500 ml

2 x 1000 ml 500 ml

Buffer WASH

300 ml

1000 ml 500 ml

3 x 1000 ml

300 ml

1000 ml 500 ml

Buffer ELU

180 ml

900 ml

2 x 900 ml

180 ml

900 ml

RNase A
(lyophilized)

10 mg

50 mg

2 x 50 mg

10 mg

50 mg

NucleoBond® Xtra Maxi columns

10

50

100

10

50

NucleoBond® Xtra Maxi column filters

10

50

100

10

50

NucleoBond® Finalizer Large

-

-

-

10

50

30 ml Syringes

-

-

-

10

50

1 ml Syringes

-

-

-

10

50

Buffer TRIS

-

-

-

15 ml

75 ml

Plastic washer

5

10

10

5

10

Protocol

1

1

1

1

1

Cat. No.




For preparation of working solutions and storage conditions see section 5.

6

MACHEREY-NAGEL – 10/2006/ Rev. 02

Plasmid DNA Purification

2

Kit specifications •

NucleoBond® Xtra kits are suitable for ultra fast purification of plasmids, cosmids, and very large constructs (P1 constructs, BACs, PACs) ranging from 3 kb up to 300 kb. For preparation of working solutions and storage conditions see section 5.

•

NucleoBond® Xtra columns are polypropylene columns containing NucleoBond® Xtra silica resin packed between two inert filter elements. The columns are available in Midi and Maxi sizes with typical maximum DNA yields of 250 µg and 1000 µg, respectively.

•

All NucleoBond® Xtra columns are resistant to organic solvents such as alcohol, chloroform, and phenol and are also suitable for buffers containing denaturing agents like formamide, urea, or common detergents like Triton X-100 or NP-40.

•

NucleoBond® Xtra silica resin can be used over a wide pH range (pH 2.5–8.5), and can remain in contact with buffers for several hours without any change in its chromatographic properties.

•

The NucleoBond® Xtra column filters are specially designed depth filters that fit into the NucleoBond Xtra columns. The filters are inserted ready-to-use in the NucleoBond® Xtra columns and allow a time-saving simultaneous clearing of bacterial lysate and loading of cleared lysate onto the NucleoBond Xtra column. Furthermore, the use of the column filters avoids the timeconsuming centrifugation step for lysate clearing.

•

The NucleoBond® Xtra column filters allow complete removal of precipitate even with large lysate volumes without clogging and avoid shearing of large DNA constructs, such as PACs or BACs by the gentle depth filter effect.

•

The NucleoBond® Xtra Midi Plus and NucleoBond® Xtra Maxi Plus kits additionally contain the NucleoBond® Finalizer and NucleoBond® Finalizer Large, respectively. These tools for a fast concentration and desalination of eluates are suitable for most plasmids and cosmids ranging from 2-50 kb with recovery efficiencies from 40-90% (depending on elution volume).

•

NucleoBond® Finalizer is a polypropylene syringe filter containing a special silica membrane. The NucleoBond® Finalizer provides a binding capacity of 500 µg, whereas the NucleoBond® Finalizer Large can hold up to 2000 µg plasmid DNA.

•

Due to the small dead volumes of NucleoBond® Finalizers the plasmid DNA can be eluted with a concentration up to 2.0 µg/µl (see section 4.11, Figures 4 and 5 for dependence of concentration on elution volume).

•

All NucleoBond® Finalizers are resistant to organic solvents such as alcohol, chloroform, and phenol and are free of endotoxins. MACHEREY-NAGEL – 10/2006/ Rev. 02

7

Plasmid DNA Purification

3

About this user manual

The following section 4 provides you with a detailed description of the NucleoBond® Xtra purification system and important information about cell growth, cell lysis, and the subsequent purification steps. Sections 5 and 6 inform you about storage, buffer preparation, and safety instructions. First-time users are strongly advised to read these chapters thoroughly before using this kit. Experienced users can directly proceed with the purification protocols (section 7) or just use the Protocol-at-a-glance for a quick reference. Each procedural step in the purification protocol is arranged like the following example taken from section 7.1: Midi 5

Maxi

Cell lysis Check lysis buffer LYS for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30-40°C until precipitate is dissolved completely. Cool buffer down to room temperature (20-25°C). Add lysis buffer LYS to the suspension. Mix gently by inverting the tube 5 times. Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension. Incubate the mixture at room temperature (20-25°C) for 5 min. Note: Increase LYS buffer volume proportionally if more than the recommended cell mass is used (see section 4.6 for information on optimal cell lysis).

8 ml

12 ml

If you are performing a Midi prep to purify plasmid DNA you will find volumes or incubation times in the white boxes. For Maxi preps please refer to the black boxes. The name of the buffer, buffer volume, incubation times, repeats or important handling steps are emphasized in bold type within the instruction. Additional notes or optional steps are printed in italic. The exclamation point marks information and hints that are essential for a successful preparation. In the example shown above you are asked to check the lysis buffer LYS prior to use and then to lyse the resuspended cell pellet in 8 ml of buffer LYS when performing a Midi prep and in 12 ml for a Maxi prep, respectively. Follow the handling instructions exactly and note the given hints for protocol alterations.

8

MACHEREY-NAGEL – 10/2006/ Rev. 02

Plasmid DNA Purification

4

NucleoBond® Xtra plasmid purification system

4.1 Basic principle The bacterial cells are lysed by an optimized set of newly formulated buffers based on the NaOH/SDS lysis method of Birnboim and Doly
. After equilibration of the NucleoBond® Xtra column together with the corresponding NucleoBond® Xtra column filter, the entire lysate is loaded by gravity flow and simultaneously cleared by the newly designed column filter. Plasmid DNA is bound to the improved NucleoBond® Xtra silica resin. After an efficient washing step the plasmid DNA is eluted, precipitated, and easily dissolved in any suitable buffer (e.g. low-salt buffer or water) for further use.

4.2 NucleoBond® Xtra anion-exchange columns NucleoBond® Xtra is a silica-based anion-exchange resin, developed by MACHEREY-NAGEL and covered under European Patent EP 0 496 822. It is developed for routine separation of different classes of nucleic acids like oligonucleotides, RNA and plasmids. NucleoBond® Xtra silica resin consists of hydrophilic, macroporous silica beads functionalized with MAE (methyl-amino-ethanol). The dense coating of this functional group provides a high overall positive charge density under acidic pH conditions that permits the negatively charged phosphate backbone of plasmid DNA to bind with high specificity (Figure 1).

Figure 1




Ionic interaction of the positively charged methyl-hydroxyethyl-amino group with the negative phosphate oxygen of the DNA backbone. In contrast to the widely used DEAE (diethylaminoethanol) group, the hydroxy group of methyl-hydroxyethyl-amin can be involved in additional hydrogen bonding interactions with the DNA.

Birnboim, H. C. and Doly, J., (1979) Nucl. Acids Res. 7, 1513-1523

MACHEREY-NAGEL – 10/2006/ Rev. 02

9

Plasmid DNA Purification Due to a specialized manufacturing process that is strictly controlled and monitored, the NucleoBond® Xtra silica beads are uniform in diameter and contain particularly large pores. These special properties allow optimized flow rates and sharp, welldefined elution profiles. NucleoBond® Xtra can separate distinct nucleic acid species from each other and from proteins, carbohydrates, and other unwanted cellular components over an exceptionally broad range of salt concentrations (Figure 2). All contaminants from proteins to RNA are washed from the column, the positive charge of the resin is neutralized by a pH shift to slightly alkaline conditions and pure plasmid DNA is eluted in a high-salt elution buffer. The purified nucleic acid products are suitable for use in the most demanding molecular biology applications, including transfection, in vitro transcription, automated or manual sequencing, cloning, hybridization, and PCR.

Figure 2

10

Elution profile of NucleoBond® Xtra silica resin at pH 7.0 The more interactions a nucleic acid can form between phosphate backbone and the positively charged resin the later it is eluted with increasing salt concentration. Large nucleic acids carry more charges than short ones, double stranded DNA more than single stranded RNA.

MACHEREY-NAGEL – 10/2006/ Rev. 02

Plasmid DNA Purification

4.3 Growth of bacterial cultures Yield and quality of plasmid DNA highly depend on the type of culture media and antibiotics, the bacterial host strain, the plasmid type, size, and copy number. For standard high-copy plasmids LB (Luria-Bertani) medium is recommended. The cell culture should be incubated at 37°C with constant shaking (200-250 rpm) preferably 12-16 h over night. Use flasks of at least three or four times the volume of the culture volume to provide a growth medium saturated with oxygen. Alternatively, rich media like 2xYT (Yeast/Tryptone), TB (Terrific Broth) or CircleGrow can be used. In this case bacteria grow faster, reach the stationary phase much earlier than in LB medium (
12 h), and higher cell masses can be reached. However, this does not necessarily yield more plasmid DNA. Overgrowing a culture might lead to a higher percentage of dead or starving cells and the resulting plasmid DNA might be partially degraded or contaminated with chromosomal DNA. To find the optimal culture conditions, the culture medium and incubation times have to be optimized for each host strain/plasmid construct combination individually. Cell cultures should be grown under antibiotic selection at all times to ensure plasmid propagation. Without this selective pressure, cells tend to lose a plasmid during cell division. Since bacteria grow much faster without the burden of a high-copy plasmid, they take over the culture rapidly and the plasmid yield goes down regardless of the cell mass. Table 1 gives information on concentrations of commonly used antibiotics.

Table 1: Information about antibiotics according to Maniatis Antibiotic

Stock solution (concentration)

Storage

Working concentration

Ampicillin

50 mg/ml in H2O

-20°C

50-100 µg/ml

Chloramphenicol

34 mg/ml in EtOH

-20°C

25-170 µg/ml

Kanamycin

10 mg/ml in H2O

-20°C

10-50 µg/ml

Streptomycin

10 mg/ml in H2O

-20°C

10-50 µg/ml

Tetracycline

5 mg/ml in EtOH

-20°C

10-50 µg/ml

The E. coli host strain mostly influences the quality of the plasmid DNA. Whereas strains like DH5 or XL1-Blue usually produce high quality super-coiled plasmid DNA, other strains like e.g. HB101 with high levels of endonuclease activity might yield lower quality plasmid giving poor results in downstream applications like enzymatic restriction or sequencing.



Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual, Cold Spring Harbor, Cold Spring, New York 1982.

MACHEREY-NAGEL – 10/2006/ Rev. 02

11

Plasmid DNA Purification The type of plasmid, especially the size and the origin of replication (ori) has a crucial influence on DNA yield. In general, the larger the plasmid or the cloned insert is, the lower is the expected DNA yield due to a lower copy number. Even a highcopy construct based on a ColE1 ori can behave like a low-copy vector in case of a large or unfavorable insert. In addition, the ori itself influences the yield by factor 10 100. Thus plasmids based on e.g. pBR322 or pACYC, cosmids or BACs are maintained at copy numbers